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Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Published on: September 5, 2019

A fluorogenic array for temporally unlimited single-molecule tracking.

Rajarshi P Ghosh^1,2,3,4, J Matthew Franklin^1,2,3,4,5, Will E Draper^1,2,3,4

¹Bioengineering, Stanford University, Stanford, CA, USA.

Nature Chemical Biology

|March 13, 2019

Summary

This summary is machine-generated.

New optical tags enable long-term intracellular tracking of single molecules. These ArrayG and ArrayD tags offer bright, fluorogenic imaging for studying molecular dynamics and chromosomal behavior in living cells.

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Last Updated: Jan 28, 2026

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Published on: September 5, 2019

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A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Published on: June 20, 2020

Area of Science:

Molecular Biology
Cell Biology
Biophysics

Background:

Intracellular tracking of single molecules is crucial for understanding cellular dynamics.
Existing optical tags face limitations in brightness, photostability, and tracking duration.
Developing novel tags is essential for advancing live-cell imaging and molecular studies.

Purpose of the Study:

To introduce and characterize three novel optical tags: ArrayG, ArrayD, and ArrayG/N.
To demonstrate the capability of these tags for long-term, high-resolution intracellular single-molecule tracking.
To explore new avenues for studying molecular dynamics and chromosomal behavior in living cells.

Main Methods:

Development of fluorogenic tags based on nanobody arrays and fluorescent protein binders (ArrayG).
Utilizing dihydrofolate reductase nanobody arrays for dual-color imaging (ArrayD).
Implementing a balanced system of binder production, photobleaching, and stochastic exchange for extended tracking.
High-speed and long-term tracking of tagged kinesins, integrins, and histones in living cells.

Main Results:

ArrayG exhibits a ~26-fold increase in brightness upon binding, enabling temporally unlimited single-molecule tracking.
High-speed tracking revealed novel dynamical features of kinesins and integrins.
ArrayG/N facilitated over 1-hour tracking of single histones, revealing Rouse polymer behavior of chromosomal loci with visco-elastic memory.
ArrayD enables dual-color imaging for simultaneous tracking of multiple molecules.

Conclusions:

The developed ArrayG, ArrayD, and ArrayG/N tags offer significant advantages in brightness, fluorogenicity, and tracking duration.
These tags provide powerful tools for investigating complex molecular dynamics and chromosomal organization in live cells.
The study opens new possibilities for advanced single-molecule imaging and quantitative cell biology research.